Stable microbubble suspensions as enhancement agents for ultrasound echography

ABSTRACT

Disclosed are injectable suspensions of gas filled microbubbles in an aqueous carrier liquid usable as contrast agents in ultrasonic echography. The suspensions comprise amphipathic compounds of which at least one may be a laminarize phospholipid as a stabiliser of the microbubbles against collapse with time and pressure. The concentration of phospholipids in the carrier liquid is below 0.01% wt but is at least equal to or above that at which phospholipid molecules are present solely at the gas microbubble-liquid interface. Also disclosed is a method of preparation of the stable suspensions of air or gas filled microbubbles.

TECHNICAL FIELD

[0001] The invention relates to injectable suspensions of gas filledmicrobubbles in an aqueous carrier comprising amphipathic compounds ofwhich at least one is a phospholipid stabilizer of the microbubblesagainst collapse with time and pressure. The phospholipid stabilizer maybe in a lamellar or laminar form. The invention also comprises a methodof making stable suspensions of microbubbles usable as contrast agentsin ultrasonic echography.

BACKGROUND OF INVENTION

[0002] Use of suspensions of gas microbubbles in a carrier liquid asefficient ultrasound reflectors is well known in the art. Thedevelopment of microbubble suspensions as echopharmaceuticals forenhancement of ultrasound imaging followed early observations that rapidintravenous injections can cause solubilized gases to come out ofsolution forming bubbles. Due to their substantial difference inacoustic impedance relative to blood, these intravascular gas bubblesare found to be excellent reflectors of ultrasound. Injecting into theblood-stream of living organisms suspensions of gas microbubbles in acarrier liquid strongly reinforces ultrasonic echography imaging, thusenhancing the visualisation of internal organs. Since imaging of organsand deep seated tissue can be crucial in establishing medical diagnosisa lot of effort is devoted to the development of stable suspensions ofhighly concentrated gas microbubbles which at the same time would besimple to prepare and administer, would contain a minimum of inactivespecies, would be capable of long storage and simple distribution. Manyattempts towards a solution which will satisfy these criteria have beenmade, however, none have provided a completely satisfactory result.

[0003] It has been known from EP-A-0 077 752 (Schering) that suspensionsof gas microbubbles can be made by mixing an aqueous solution of asurfactant with a solution of a viscosity enhancer as a stabilizer. Thegas bubbles are introduced into the mixture by forcing the mixture ofreagents and air through a small aperture. A suspension of CO₂microbubbles may be obtained by addition of an acid to a mixtureobtained from a solution containing a surfactant and sodium bicarbonateand a solution of the viscosity enhancer. Mixing the components however,is to be carried out just before use and the solution is to beconsumed/injected immediately upon preparation. The disclosedsurfactants (tensides) comprise lecithins; esters and ethers of fattyacids and fatty alcohols with polyoxyethylene and polyoxyethylatedpolyols like sorbitol, glycols and glycerol, cholesterol; andpolyoxy-ethylene-polyoxypropylene polymers. Disclosed concentration oftensides in the suspension is between 0.01% and 10% wt and a preferredrange is claimed to be between 0.5% to 5%. The viscosity enhancing andstabilizing compounds include for instance mono- and polysaccharides(glucose, lactose, sucrose, dextran, sorbitol): polyols, e.g. glycerol,polyglycols; and polypeptides like proteins, gelatin, oxypolygelatin,plasma protein and the like. The total amount of viscosity enhancingagent is limited to 0.5 and 50%. Use of polyoxypropylene-polyoxyethylenepolymers (eg. Pluronic® F-68) as viscosity enhancing agent has also beendisclosed. In the preferred example, equivalent volumes of tenside, a0.5% by weight aqueous solution of Pluronic® F-68 (apolyoxypropylene-polyoxyethylene copolymer), and the viscosity enhancer(a 10% lactose solution) are vigorously shaken together under sterileconditions to provide a suspension of microbubbles. The suspensionobtained lasted over 2 minutes and contained close to 50% of bubbleswith a size below 50 μm. According to the document up to 50% ofsurfactants and/or viscosity enhancing agents may be employed, however,specific examples use between 1% and 4% of Pluronic® F-68.

[0004] Easy-to-produce aqueous suspensions usable as imaging agents inultrasonic echography are disclosed in WO-91/15244 (Schneider et. al.).The suspensions contain film forming surfactants in laminar and/orlamellar form and, optionally, hydrophilic stabilzers. The laminarizedsurfactants can be in the form of liposomes i.e. microscopic vesicles,generally spherically shaped. These vesicles are usually formed of oneor more concentrically arranged bi-molecular layers of amphipathiccompounds i.e. compounds with a hydrophilic and a hydrophobic moieties.The molecules in the bilayers are organised so that the hydrophobicmoieties are in facing relationship, the hydrophilic moieties pointingtoward the water phase. The suspensions are obtained by exposing thelaminarized surfactants to air or a gas prior to or after admixing withan aqueous phase. Conversion of film forming surfactants into lamellarform is carried out according to various liposome forming techniquesincluding high pressure homogenisation or sonication under acoustic orultrasonic frequencies. The concentration of phospholipids claimed isbetween 0.01% and 20% and the concentration of microbubbles is between10⁸ and 10⁹ bubbles/ml. The microbubble suspensions remained stable formonths. The concentration of phospholipids in Example 1 is 0.5%.

[0005] An attempt toward a stable echogenic suspension is disclosed inWO-92/11873 (Beller et al.). Aqueous preparations designed to absorb andstabilise microbubbles for use as an echographic contrasting agent aremade with polyoxyethylene/polyoxypropylene polymers and negativelycharged phospholipids such as phosphatidylglycerol,phosphatidylinositol, phosphatidylethanolamine, phosphatidylserine aswell as their lysoforms. The concentration range of phospholipids in thepreparations may be between 0.01% and 5% by volume or weight, however,preparations with 1% of dipalmitoylphosphatidyl glycerol (DPPG) arespecifically disclosed and claimed. In addition to the negativelycharged phospholipids the compositions must contain between 0.1% and 10%of polymeric material (Pluronic® F-68). The total amount of solutes inthe preparations is between 5.1% and 10.4%. The concentration of themicrobubbles is not reported, however, according to the results given itmay be estimated to be about 10⁷ bubbles/ml. The stability of thesuspensions is reported to be better than that of EP-A-0 077 752.

[0006] Although the prior art compositions have merit, they still sufferseveral drawbacks which hamper their practical use. Firstly, some priorart compositions have relatively short life spans and secondly, theyhave a relatively low initial bubble count e.g. between 10⁴ and 10⁵bubbles/ml. This makes reproducibility and analysis of echographic testsmade with such compositions fairly difficult. In addition, sometechniques produce bubbles in a wide range of diameters (up to 50 μm)which prevents their use as echographic agents in certain applications(e.g. echography of the left heart).

[0007] The need for stable formulations of microbubbles which willresist pressure variations in the blood streams and have a good shelflife is further amplified by poor stability of some of thestate-of-the-art compositions. Microbubble formulations whosedistribution and storage would not present problems are particularlyimportant.

[0008] Another drawback is that many of the heretofore knowncompositions contain a high amount of different solutes such aspolymers, phospholipids, electrolytes, and other which render theirpractical use more and more difficult. For example, it is known that useof polyoxyethylene/polyoxypropylene polymers (Pluronic®) with particularpatients may cause unpleasant side effects (see for instance G. M.Vercellotti et. al. Blood (1982) 59, 1299). Preparations with a highphospholipid content in certain cases may also be undesirable. In anyevent, compositions with a high degree of various solutes areadministered reluctantly and their wide spread use is becomingconsidered to be undesirable. In fact, the trend in the pharmaceuticalindustry is to reduce concentrations of active and inactive ingredientsin various medical or pharmaceutical formulations to their lowestpossible levels and eliminate from the preparations everything that isnot necessary. Finding alternative methods and formulating moreeffective compositions continues to be important. This is particularlyso with microbubble suspensions used in echography since here theingredients have no curative effect and should lead to the leastpossible after consequences. However, as stated above, the state of theart preparations with typical concentrations in the range of 1% and 4%by weight and the teachings of prior art discourage use of reducedamounts of phospholipids and other non-phospholipid additives. Thereason for the discouragement is most probably hidden in the fact thatin the course of the routine experimentation further reduction inconcentration of the ingredients never produced suspensions which werestable enough to have any practical use or encourage further tinkeringin the lower end of the known range.

SUMMARY OF THE INVENTION

[0009] The present invention is based on the unexpected finding thatvery stable suspensions of a gas filled microbubbles comprising at least10⁷ microbubbles per millilitre may be obtained using phospholipids asstabilizers even if very low concentrations thereof are employed. Thesuspensions usable as contrasting agents in ultrasonic echography areobtained by suspending in an aqueous carrier at least one phospholipidas a stabiliser of the microbubbles against collapse with time andpressure, the concentration of the phospholipids being below 0.01% wt.but equal to or higher than that at which the phospholipid molecules arepresent solely at the gas microbubble-liquid interface.

[0010] It was quite unexpected to discover that as negligible amounts ofthe phospholipid surfactants involved here (used alone or with arelatively small proportions of other amphiphiles) can so effectivelystabilize microbubbles. It is postulated that, in the presence of otheramphipathic compounds (such as Pluronic®) the mutual cohesion betweenstabilizer molecules is decreased and formation of monomolecularphospholipid films is inhibited. However, in the absence of largeamounts of other amphiphilic agents, the unhindered intermolecularbinding forces (electrostatic interaction or hydrogen bonding) betweenphospholipid molecules are sufficient to ensure formation of stablefilm-like structures stabilizing the bubbles against collapse orcoalescence.

[0011] According to the invention, suspensions of high microbubbleconcentration, high stability, long storage capacity and ease ofpreparation may be obtained even if the concentrations of surfactantsand other additives in the suspensions are kept well below the levelsused in the state-of-the-art formulations. The amount of phospholipidsused in the compositions of the invention may be as low as about thatonly necessary for formation of a single monolayer of the surfactantaround the gas microbubbles while the concentration of the bubbles inthe suspension is maintained above 10⁷ microbubbles per millilitre. Inthe present invention, microbubbles with a liposome-like double layer ofsurfactant (gas filled liposomes) are not likely to exist and have notbeen observed.

[0012] Suspensions with high microbubble concentrations e.g. between 10⁹and 10¹⁰ bubbles/ml of relatively high stability and long storagecapacity may be prepared even if the concentration of the phospholipidsurfactants are kept well below the levels known in the art. Suspensionswith as little as 1 μg of phospholipids per ml may be prepared as longas the amount of the surfactants used is not below that which isnecessary for formation of a single monolayer of the lipids around thegas microbubbles and as long as they are produced according to one ofthe methods herein disclosed.

[0013] Calculations have shown that for bubble concentrations of 10⁸bubbles/ml depending on the size distribution of the microbubbles thisconcentration may be as low as 1 μg/ml or 0.0001%, however, thephospholipid concentrations between 0.0002% and up to 0.01% arepreferred. More preferably the concentration of the phospholipids in thestable suspensions of microbubbles of the invention is between 0.001%and 0.009%. Although further reduction of the amount of phospholipids inthe suspension is possible, suspensions prepared with less than 0.0001%wt. are unstable, their total bubble count is low and their echographicresponse upon injection is not satisfactory. On the other hand,suspensions prepared with more than 0.01% of phospholipids uponinjection do not perform better i.e. their stability and echographicresponse do not further improve with the concentration. Thus, the higherconcentrations may only increase the probability of undesirable sideeffects as set out in the discussion of the prior art. It is tentativelypostulated that only the segments of the surfactants which are in thelamellar or laminar form can effectively release molecules organizedproperly to stabilize the bubbles. This may explain why theconcentration of the surfactant may be so low without impairing thestability of the gas bubbles.

[0014] The suspensions of the invention offer important advantages overthe compositions of the prior art not only because of the lowphospholipid content but also because the total amount of injectedsolutes i.e. lipids and/or synthetic polymers and other additives isbetween 1,000 and 50,000 times lower than heretofore. This is achievedwithout any loss of microbubble concentration i.e. echogenicity orstability of the product. In addition to the very low concentration ofsolutes, the invention provides suspensions which may contain only themicrobubbles whose contribution to the echographic signal is relativelysignificant i.e. suspensions which are free of any microbubbles which donot actively participate in the imaging process.

[0015] Needless to say that with such low concentrations of solutes inthe injectable composition of the invention probability of undesirableside effects is greatly reduced and elimination of the injected agent issignificantly improved.

[0016] The microbubble suspensions with low phospholipid content of theinvention may be prepared from the film forming phospholipids whosestructure has been modified in a convenient manner e.g. by freeze-dryingor spray-drying solutions of the crude phospholipids in a suitablesolvent. Prior to formation of the suspension by dispersion in anaqueous carrier the freeze dried or spray dried phospholipid powders arecontacted with air or another gas. When contacted with the aqueouscarrier the powdered phospholipids whose structure has been disruptedwill form lamellarized or laminarized segments which will stabilise themicrobubbles of the gas dispersed therein. Conveniently, the suspensionswith low phospholipid content of the invention may also be prepared withphospholipids which were lamellarized or laminarized prior to theircontacting with air or another gas. Hence, contacting the phospholipidswith air or another gas may be carried out when the phospholipids are ina dry powder form or in the form of a dispersion of laminarizedphospholipids in the aqueous carrier.

[0017] The term lamellar or laminar form indicates that the surfactantsare in the form of thin films or sheets involving one or more molecularlayers. In this form, the surfactant molecules organize in structuressimilar to that existing in liposome vesicles. As described inWO-A-91/15244 conversion of film forming surfactants into lamellar formcan easily be done by any liposome forming method for instance by highpressure homogenisation or by sonication under acoustical or ultrasonicfrequencies. The conversion into lamellar form may also be performed bycoating microparticles (10 μm or less) of a hydrosoluble carrier solid(NaCl, sucrose, lactose or other carbohydrates) with a phospholipid withsubsequent dissolution of the coated carrier in an aqueous phase.Similarly, insoluble particles, e.g. glass or resin microbeads may becoated by moistening in a solution of a phospholipid in an organicsolvent following by evaporation of the solvent. The lipid coatedmicrobeads are thereafter contacted with an aqueous carrier phase,whereby liposomic vesicles will form in the carrier phase. Also,phospholipids can be lamellarized by heating slightly above criticaltemperature (Tc) and gentle stirring. The critical temperature is thetemperature of gel-to-liquid transition of the phospholipids.

[0018] Practically, to produce the low phospholipid content suspensionsof microbubbles according to the invention, one may start with liposomesuspensions or solutions prepared by any known technique as long as theliposomic vesicles are “unloaded”, i.e. they do not have encapsulatedtherein any foreign material but the aqueous phase of the solutionitself.

[0019] The introduction of air or gas into a liposome solution can beeffected by usual means, injection i.e. forcing air or gas through tinyorifices into the liposome solution, or simply dissolving the gas in thesolution by applying pressure and then suddenly releasing the pressure.Another way is to agitate or sonicate the liposome solution in thepresence of air or another physiologically acceptable gas. Also one cangenerate the formation of a gas within the solution of liposomes itself,for instance by a gas releasing chemical reaction, e.g. decomposing adissolved carbonate or bicarbonate by acid.

[0020] When laminarized surfactants are suspended in an aqueous liquidcarrier and air or another gas is introduced to provide microbubbles, itis thought that the microbubbles become progressively surrounded andstabilised by a monomolecular layer of surfactant molecules and not abilayer as in the case of liposome vesicles. This structuralrearrangement of the surfactant molecules can be activated mechanically(agitation) or thermally. The required energy is lower in the presenceof cohesion releasing agents, such as Pluronic®. On the other hand,presence of the cohesion releasing agents in the microbubbleformulations reduces the natural affinity between phospholipid moleculeshaving as a direct consequence a reduced stability of the microbubblesto external pressures (e.g. above 20-30 Torr).

[0021] As already mentioned, to prepare the low phospholipid contentsuspensions of the invention, in place of phospholipid solutions, onemay start with dry phospholipids which may or may not be lamellarized.When lamellarized, such phospholipids can be obtained for instance bydehydrating liposomes, i.e. liposomes which have been prepared normallyby means of conventional techniques in the form of aqueous solutions andthereafter dehydrated by usual means. One of the methods for dehydratingliposomes is freeze-drying (lyophilization), i.e. the liposome solution,preferably containing hydrophilic compounds, is frozen and dried byevaporation (sublimation) under reduced pressure.

[0022] In another approach, non-lamellarized or non-laminarizedphospholipids may be obtained by dissolving the phospholipid in anorganic solvent and drying the solution without going through liposomeformation. In other words, this can be done by dissolving thephospholipids in a suitable organic solvent together with a hydrophilicstabiliser substance e.g. a polymer like PVP, PVA, PEG, etc. or acompound soluble both in the organic solvent and water and freeze-dryingor spray-drying the solution. Further examples of the hydrophilicstabiliser compounds soluble in water and the organic solvent are malicacid, glycolic acid, maltol and the like. Any suitable organic solventmay be used as long as its boiling point is sufficiently low and itsmelting point is sufficiently high to facilitate subsequent drying.Typical organic solvents would be for instance dioxane, cyclohexanol,tertiary butanol, tetrachlorodifluoro ethylene (C₂Cl₄F₂) or2-methyl-2-butanol however, tertiary butanol, 2-methyl-2-butanol andC₂Cl₄F₂ are preferred. In this variant the criteria used for selectionof the hydrophilic stabiliser is its solubility in the organic solventof choice. The suspensions of microbubbles are produced from suchpowders using the same steps as with powders of the laminarizedphospholipids.

[0023] Similarly, prior to effecting the freeze-drying ofpre-lamellarized or pre-laminarized phospholipid solutions, ahydrophilic stabiliser compound is dissolved in the solution. However,here the choice of the hydrophilic stabilisers is much greater since acarbohydrate like lactose or sucrose as well as a hydrophilic polymerlike dextran, starch, PVP, PVA PEG and the like may be used. This isuseful in the present invention since such hydrophilic compounds alsoaid in homogenising the microbubbles size distribution and enhancestability under storage. Actually making very dilute aqueous solutions(0.0001-0.01% by weight) of freeze-dried phospholipids stabilised with,for instance, a 10:1 to 1000:1 weight ratio of polyethyleneglycol tolipid enables to produce aqueous microbubbles suspensions counting10⁹-10¹⁰ bubbles/ml (size distribution mainly 0.5-10 μm) which arestable, without significant observable change, even when stored forprolonged periods. This is obtained by simple dissolution of theair-stored dried laminarized phospholipids without shaking or anyviolent agitation. The freeze-drying technique under reduced pressure isvery useful because it permits, restoration of the pressure above thedried powders with any physiologically acceptable gas, i.e. nitrogen,CO₂, argon, methane, freons, SF₆, CF₄, etc., whereby after redispersionof the phospholipids processed under such conditions suspensions ofmicrobubbles containing the above gases are obtained.

[0024] It has been found that the surfactants which are convenient inthis invention can be selected from amphipathic compounds capable offorming stable films in the presence of water and gases. The preferredsurfactants include the lecithins (phosphatidylcholine) and otherphospholipids, inter alia phosphatidic acid (PA), phosphatidylinositolphosphatidylethanolamine (PE), phosphatidylserine (PS),phosphatidylglycerol (PG), cardiolipin (CL), sphingomyelins. Examples ofsuitable phospholipids are natural or synthetic lecithins, such as eggor soya bean lecithin, or saturated synthetic lecithins, such as,dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine,distearoylphosphatidylcholine or diarachidoylphosphatidylcholine orunsaturated synthetic lecithins, such as dioleylphosphatidyl choline ordilinoleylphosphatidylcholine, with saturated lecithins being preferred.

[0025] Additives like cholesterol and other substances can be added toone or more of the foregoing lipids in proportions ranging from zero to50% by weight. Such additives may include other non-phospholipidsurfactants that can be used in admixture with the film formingsurfactants and most of which are known. For instance, compounds likepolyoxypropylene glycol and polyoxyethylene glycol as well as variouscopolymers thereof, phosphatidylglycerol, phosphatidic acid,dicetylphosphate, fatty acids, ergosterol, phytosterol, sitosterol,lanosterol, tocopherol, propyl gallate, ascorbyl palmitate and butylatedhydroxy-toluene. The amount of these non-film forming surfactants areusually up to 50% by weight of the total amount of surfactants butpreferably between 0 and 30%. Again this means that the concentration ofthe various additives in the low phospholipid content suspensions of theinvention are in the range of 0-0.05% which is more than one hundredtimes less than in the compositions known so far.

[0026] It should also be mentioned that another feature of thesuspensions of the invention is a relatively “high” gas entrappingcapacity of the microbubbles i.e. high ratio between the amount of thesurfactant and the total amount of the entrapped gas. Hence, withsuspensions in which the microbubbles have sizes in the 1 to 5 μm range,it is tentatively estimated that the weight ratio of phospholipidspresent at the gas bubble-liquid interface to the volume of entrappedgas under standard conditions is between 0.1 mg/ml and 100 mg/ml.

[0027] In practice all injectable compositions should also be as far aspossible isotonic with blood. Hence, before injection, small amounts ofisotonic agents may also be added to the suspensions of the invention.The isotonic agents are physiological solutions commonly used inmedicine and they comprise aqueous saline solution (0.9% NaCl). 2.6%glycerol solution, 5% dextrose solution, etc.

[0028] The invention further concerns a method of making stablesuspensions of microbubbles according to claim 1 usable as contrastagents in ultrasonic echography. Basically, the method comprisesadapting the concentration of the phospholipids in the suspension ofmicrobubbles stabilized by said phospholipids to a selected value withinthe limits set forth in the claims. Usually, one will start with amicrobubble suspension containing more phospholipids than the valuedesired and one will reduce the amount of said phospholipids relativelyto the volume of gas or air entrapped in the microbubble, withoutsubstantially reducing the count of echogenerating bubbles. This can bedone, for instance, by removing portions of the carrier liquidcontaining phospholipids not directly involved at the air/liquidinterface and diluting the suspension with more fresh carrier liquid.For doing this, one may create within the suspension region (a) wherethe echogenerating bubbles will gather and region (b) where said bubblesare strongly diluted. Then the liquid in region (b) can be withdrawn byseparation by usual means (decantation, siphoning, etc) and a comparablevolume of fresh carrier liquid is supplied for replenishment to thesuspension. This operation can be repeated one or more times, wherebythe content in phospholipids not directly involved in stabilizing thebubbles will be progressively reduced.

[0029] It is generally not desirable to achieve complete removal of thephospholipid molecules not present at the bubble gas/liquid interface assome unbalance from equilibrium may result, i.e. if the depletion isadvanced too far, some surfactant molecules at the gas/liquid interfacemay be set free with consequent bubble destabilization. Experiments haveshown that the concentration of phospholipids in the carrier liquid maybe decreased down to within the neighborhood of the lower limit setforth in the claims without significant changes in properties andadverse effects. This means that, actually, the optimal phospholipidconcentration (within the given limits) will be rather dictated by thetype of application i.e. if relatively high phospholipid concentrationsare admissible, the ideal concentration value will be near the upperlimit of the range. On the other hand, if depending on the condition ofthe patient to be diagnosed, the absolute value of phospholipids must befurther reduced, this can be done without adverse effects regardingmicrobubble count and echogenic efficiency.

[0030] An embodiment of the method comprises selecting a film formingsurfactant and optionally converting it into lamellar form using one ofthe methods known in the art or disclosed hereinbefore. The surfactantis then contacted with air or another gas and admixed with an aqueousliquid carrier in a closed container whereby a suspension ofmicrobubbles will form. The suspension is allowed to stand for a whileand a layer of gas filled microbubbles formed is left to rise to the topof the container. The lower part of the mother liquor is then removedand the supernatant layer of microbubbles washed with an aqueoussolution saturated with the gas used in preparation of the microbubbles.This washing can be repeated several times until substantially allunused or free surfactant molecules are removed. Unused or freemolecules means all surfactant molecules that do not participate information of the stabilising monomolecular layer around the gasmicrobubbles.

[0031] In addition to providing the low phospholipid contentsuspensions, the washing technique offers an additional advantage inthat it allows further purification of the suspensions of the invention,i.e. by removal of all or almost all microbubbles whose contribution tothe echographic response of the injected suspension is relativelyinsignificant. The purification thus provides suspensions comprisingonly positively selected microbubbles, i.e. the microbubbles which uponinjection will participate equally in the reflection of echographicsignals. This leads to suspensions containing not only a very lowconcentration of phospholipids and other additives, but free from anymicrobubbles which do not actively participate in the imaging process.

[0032] In a variant of the method, the surfactant which optionally maybe in lamellar form, is admixed with the aqueous liquid carrier prior tocontacting with air or another gas.

BRIEF DESCRIPTION OF DRAWINGS

[0033]FIG. 1 is graphical presentation of echographic responses as afunction of the microbubble concentration for a freshly preparedsuspension according to the invention.

[0034] Suspensions and the method of making low phospholipid contentsuspensions of the invention will be further illustrated by thefollowing examples:

EXAMPLE 1

[0035] Multilamellar vesicles (MLVs) were prepared by dissolving 240 mgof diarachidoylphosphatidylcholine (DAPC, from Avanti Polar Lipids) and10 mg of dipalmitoyl-phosphatidic acid (DPPA acid form, from AvantiPolar Lipids) in 50 ml of hexane/ethanol (8/2, v/v) then evaporating thesolvents to dryness in a round-bottomed flask using a rotary evaporator.The residual lipid film was dried in a vacuum dessicator. After additionof water (5 ml), the suspension was incubated at 90° C. for 30 minutesunder agitation. The resulting MLVs were extruded at 85° C. through a0.8 μm polycarbonate filter (Nuclepore®). 2.6 ml of the resulting MLVpreparation were added to 47.4 ml of a 167 mg/ml solution of de-tran10'000 MW (Fluka) in water. The resulting solution was thoroughly mixed,transferred in a 500 ml round-bottom flask, frozen at −45° C. andlyophilised under 0.1 Torr. Complete sublimation of the ice was obtainedovernight. Thereafter, air pressure was restored in the evacuatedcontainer. Various amounts of the resulting powder were introduced inglass vials (see table) and the vials were closed with rubber stoppers.Vacuum was applied via a needle through the stopper and the air removedfrom vials. Upon evacuation of air the powder was exposed to sulfurhexafluoride gas SF₆.

[0036] Bubble suspensions were obtained by injecting in each vial 10 mlof a 3% glycerol solution in water (through the stopper) followed bygentle mixing. The resulting microbubble suspensions were counted usinga hemacytometer. The mean bubble size (in volume) was 2.2 μm. Dry weightPhospholipid conc. Concentration (mg/ml) (μg per ml) (bubbles/ml) 0.5 89.0 × 10⁶ 1 16 1.3 × 10⁷ 5 81 7.0 × 10⁷ 10 161 1.4 × 10⁸

[0037] Preparations were injected to rabbits (via the jugular vein) aswell as minipigs (via the ear vein) at a dose of 1 ml/5 kg. In vivoechographic measurements were performed using an Acuson XP128 ultrasoundsystem (Acuson Corp. USA) and a 7 MHz sector transducer. The animalswere anaesthetised and the transducer was positioned and then fixed inplace on the left side of the chest providing a view of the right andleft ventricles of the heart in the case of rabbit and a longitudinalfour-chamber view in the case of the minipig. The preparation containing0.5 mg/ml dry weight gave slight opacification of the right as well asthe left ventricle in rabbits and in minipigs. The opacification,however, was superior with the 1.5 and 10 mg/ml preparations.

EXAMPLE 2

[0038] Lyophilisates were prepared as described in Example 1 with air(instead of SF₆) in the gas phase. The lyophilisates were then suspendedin 0.9% saline (instead of a 3% glycerol solution). Similar bubbleconcentrations were obtained. However, after injection in the rabbit orthe minipig the persistence of the effect was shorter e.g. 10-20 sinstead of 120 s. Moreover, in the mini pig the opacification of theleft ventricle was poor even with the 10 mg/ml preparation.

EXAMPLE 3

[0039] MLV liposomes were prepared as described in Example 1 using 240mg of DAPC and 10 mg of DPPA (molar ratio 95:5). Two millilitres of thispreparation were added to 20 ml of a polyethyleneglycol (PEG 2'000)solution (82.5 mg/ml). After mixing for 10 min at room temperature, theresulting solution was frozen during 5 min at −45° C. and lyophilisedduring 5 hours at 0.2 mbar. The powder obtained (1.6 g) was transferredinto a glass vial equipped with a rubber stopper. The powder was exposedto SF₆ (as described in Example 1) and then dissolved in 20 ml ofdistilled water. The suspension obtained showed a bubble concentrationof 5×10⁹ bubbles per ml with a median diameter in volume of 5.5 μm. Thissuspension was introduced into a 20 ml syringe, the syringe was closedand left in the horizontal position for 24 hours. A white layer ofbubbles could be seen on the top of solution in the syringe. Most of theliquid phase (˜16-18 ml) was evacuated while the syringe was maintainedin the horizontal position and an equivalent volume of fresh,SF₆-saturated, water was introduced. The syringe was then shaken for awhile in order to homogenise the bubbles in the aqueous phase. A seconddecantation was performed under the same conditions after 8 hoursfollowed by three further decantations performed in four hour intervals.The final bubble phase (batch P145) was suspended in 3 ml of distilledwater. It contained 1.8×10⁹ bubbles per ml with a median diameter involume of 6.2 μm. An aliquot of this suspension (2 ml) was lyophilisedduring 6 hours at 0.2 mbar. The resulting powder was dissolved in 0.2 mlof tetrahydrofuran/water (9/1 v/v) and the phospholipids present in thissolution were analysed by HPLC using a light scattering detector. Thissolution contained 0.7 mg DAPC per ml thus corresponding to 3.9 μg ofphospholipids per 10⁸ bubbles. A Coulter counter analysis of the actualbubble size distribution in batch P145 gave a total surface of 4.6×10⁷μm² per 10⁸ bubbles. Assuming that one molecule of DAPC will occupy asurface of 50 Å², one can calculate that 1.3 μg of DAPC per 10⁸ bubbleswould be necessary to form a monolayer of phospholipids around eachbubble. The suspension P145 was than left at 4° C. and the concentrationof gas bubbles measured on a regular basis. After 10 days, the productlooked as good as after its preparation and still contained 1-1.2×10⁹bubbles per ml. The exceptional stability was found very surprisingconsidering the extremely low amount of phospholipids in the suspension.

[0040] The experiment described above was repeated on a second batch ofmicrobubbles using a shorter decantation time in order to collectpreferably larger bubbles (batch P132). The median diameter in volumeobtained was 8.8 μm and the total surface determined with the Coultercounter was 22×10⁸ μm² per 10⁸ bubbles. The calculation showed that 6 μgDAPC for 10⁸ bubbles would be necessary to cover this bubble populationwith a monolayer of DAPC. The actual amount of DAPC determined by HPLCwas 20 μg per 10⁸ bubbles. Taking into account the difficulty ofobtaining precise estimates of the total surface of the bubblepopulation, it appears that within the experimental error, the resultsobtained are consistent with coverage of the microbubbles with onephospholipid layer.

[0041] Echographic measurements performed with different washed bubblepreparations showed that upon separation the lower phase gives a muchweaker echographic signal than the upper phase or a freshly preparedsample. On a first sight this seemed normal as the white layer on thetop of the syringe contained the majority of the gas microbubblesanyway. However, as shown in FIG. 1 the bubble count showed asurprisingly high microbubble population in the lower layer too. Onlyupon Coulter measurement it became apparent that the microbubbles had asize below 0.5 μm, which indicates that with small bubbles even when inhigh concentration, there is no adequate reflection of the ultrasoundsignal.

[0042] A four fold dilution of the preparation P132 in a 3% glycerolsolution was injected in the minipig (0.2 ml/kg). The preparation ofwashed bubbles containing 2.5×10⁷ bubbles per ml and 5 μg ofphospholipids per ml provided excellent opacification in the left andright ventricle with outstanding endocardial border delineation. Goodopacification was also obtained by injecting to a minipig an aliquot ofpreparation P145 (diluted in 3% glycerol) corresponding to 0.2 μg ofphospholipids per kg. Contrast was even detectable in the left ventricleafter injection of 0.02 μg/kg. Furthermore, in the renal artery theexistence of a contrast effect could be detected by pulsed Doppler atphospholipid doses as low as 0.005 μg/kg.

[0043] It follows that as long as the laminarized phospholipids arearranged in a single monolayer around the gas microbubbles thesuspensions produced will have adequate stability. Thus providing anexplanation for the present unexpected finding and demonstrating thatthe amount of phospholipids does not have to be greater than thatrequired for formation of a monolayer around the microbubbles present inthe suspension.

EXAMPLE 4

[0044] A solution containing 48 mg of DAPC and 2 mg of DPPA inhexane/ethanol 8/2 (v/v) was prepared and the solvent evaporated todryness (as described in Example 1). 5 mg of the resulting powder and375 mg of polyethyleneglycol were dissolved in 5 g of tert-butanol at60° C. The clear solution was then rapidly cooled to −45° C. andlyophilised. 80 mg of the lyophilisate was introduced in a glass vialand the powder exposed to SF₆ (see Example 1). A 3% glycerol solution(10 ml) was then introduced in the vial and the lyophilisate dissolvedby gentle swirling. The resulting suspension had 1.5×10⁸ bubbles per mlwith a median diameter (in volume) of 9.5 μm. This solution was injectedto a rabbit providing outstanding views of the right and left ventricle.Even a ten fold dilution of this suspension showed strong contrastenhancement.

EXAMPLE 5

[0045] The procedure of Example 4 was repeated except that the initialdissolution of the phospholipids in hexane/ethanol solution was omitted.In other words, crude phospholipids were dissolved, together withpolyethylene glycol in tertiary butanol and the solution wasfreeze-dried; thereafter, the residue was suspended in water. Severalphospholipids and combinations of phospholipids with other lipids wereinvestigated in these experiments. In the results shown in the nexttable the phospholipids were dissolved in a tertiary butanol solutioncontaining 100 mg/ml of PEG 2'000. The residues obtained after freezedrying were saturated with SF₆ (see Example 1), then dissolved indistilled water at a concentration of 100 mg dry weight per ml. Lipidmixture Conc. in tert- Bubble conc. Median diam (weight ratio)butanol(mg/ml) (× 10⁹/ml) (μm) DSPC 2 1.3 10 DAPC/DPPG (100/4) 2 3.8 7DSPC/Chol (2/1) 6 0.1 40 DAPC/Plur F68 (2/1) 6 0.9 15 DAPC/Palm. ac.(60/1) 2 0.6 11 DAPC/DPPA (100/4) 1 2.6 8 DAPC/Chol/DPPA (8/1/1) 8 1.219 DAPC/DPPA (100/4)* 5 2.4 18

[0046] In all cases the suspensions obtained showed high microbubbleconcentrations indicating that the initial conversion of phospholipidsinto liposomes was not necessary. These suspensions were diluted in 0.15M NaCl and injected to minipigs as described in Example 3. In all casesoutstanding opacification of the right and left ventricles as well asgood delineation of the endocardial border were obtained at doses of10-50 μg of lipids per kg body weight or less.

EXAMPLE 6

[0047] PEG-2000 (2 g), DAPC (9.6 mg) and DPPA (0.4 mg) were dissolved in20 ml of tertiary butanol and the solution was freeze dried overnight at0.2 mbar. The powder obtained was exposed to SF₆ and then dissolved in20 ml of distilled water. The suspension containing 1.4×10⁹ bubbles perml (as determined by hemacytometry) was introduced into a 20 ml syringe,which was closed and left in horizontal position for 16 hours. A whitelayer of bubbles could be seen on top of the solution. The lower phase(16-18 ml) was discarded while maintaining the syringe horizontally. Anequivalent volume of fresh SF₆-saturated distilled water was aspiratedin the syringe and the bubbles were homogenised in the aqueous phase byagitation. Two different populations of microbubbles i.e. large-sizedand medium-sized were obtained by repeated decantations over shortperiods of time, the large bubbles being collected after only 10-15 minof decantation and the medium sized bubbles being collected after 30-45min. These decantations were repeated 10 times in order to obtain narrowbubble size distributions for the two types of populations and toeliminate all phospholipids which were not associated with themicrobubbles. All phases containing large bubbles were pooled(“large-sized bubbles”). Similarly the fractions containing medium sizedbubbles were combined (“medium-sized bubbles”). Aliquots of the twobubble populations were lyophilised and then analysed by HPLC in orderto determine the amount of phospholipids present in each fraction. Thelarge-sized bubble fraction contained 2.5×10⁷ bubbles per ml with amedian diameter in number of 11.3 μm and 13.7 μg phospholipids per 10⁷bubbles. This result is in excellent agreement with the theoreticalamount, 11.5 μg per 10⁷ bubbles, calculated assuming a monolayer ofphospholipids around each bubble and a surface of 50 Å per phospholipidmolecule. The medium-sized bubble fraction contained 8.8×10⁸ bubbles perml with a median diameter in number of 3.1 μm and 1.6 μg phospholipidsper 10⁷ bubbles. The latter value is again in excellent agreement withthe theoretical amount, 1.35 μg per 10⁷ bubbles. These results furtherindicate that the stability of the microbubble suspensions hereindisclosed is most probably due to formation of phospholipid monolayersaround the microbubbles.

1. An injectable suspension of gas filled microbubbles in an aqueouscarrier liquid, usable as contrast agent in ultrasonic echography,comprising at least 10⁷ microbubbles per millilitre and amphipathiccompounds at least one of which is a phospholipid stabilizer of themicrobubbles against collapse, characterized in that the concentrationof the phospholipids in the carrier liquid is below 0.01% by weightwhile being equal to or above that at which the phospholipid moleculesare present solely at the gas microbubble-liquid interface.
 2. Theinjectable suspension of claim 1, in which the concentration ofmicrobubbles per millilitre is between 10⁸ and 10¹⁰.
 3. The injectablesuspension of claim 1, in which the concentration of phospholipids isabove 0.00013% wt.
 4. The injectable suspension of any preceding claims,in which the liquid carrier further comprises water soluble poly- andoligosaccharides, sugars and hydrophilic polymers such as polyethyleneglycols as stabilizers.
 5. The injectable suspension of any precedingclaim, in which the phospholipids are at least partially in lamellar orlaminar form and are selected from lecithins such as phosphatidic acid,phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine,phosphatidylglycerol phosphatidylinositol, cardiolipin andsphingomyelin.
 6. The injectable suspension of claim 4 or 5, furthercontaining substances affecting the properties of phospholipids selectedfrom phosphatidylglycerol, phosphatidic acid, dicetylphosphate,cholesterol, ergosterol, phytosterol, sitosterol, lanosterol,tocopherol, propylgallate, ascorbyl palmitate and butylatedhydroxytoluene.
 7. The injectable suspension of claim 1, 2 or 3, inwhich the phospholipids are in the form of powders obtained byfreeze-drying or spray-drying.
 8. The injectable suspension of claim 1,containing about 10⁸-10⁹ microbubbles per millilitre with themicrobubble size between 0.5-10 μm showing little or no variation understorage.
 9. The injectable suspension of claim 1, in which the liquidcarrier further comprises up to 50% by weight non-laminar surfactantsselected from fatty acids, esters and ethers of fatty acids and alcoholswith polyols such as polyalkylene glycols, polyalkylenated sugars andother carbohydrates, and polyalkylenated glycerol.
 10. The injectablesuspension of any preceding claim, in which the microbubbles are filledwith SF₆, CF₄, freons or air.
 11. A method of making suspensions of airor gas filled microbubbles comprising selecting at least one filmforming surfactant, converting the surfactant into a powder, contactingthe powder with air or another gas and admixing the powder surfactantwith an aqueous liquid carrier to form said suspension, characterised byintroducing the suspension into a container, forming a layer of the gasfilled microbubbles in the upper part of the container, separating thelayer of the microbubbles formed, and washing the microbubbles with anaqueous solution saturated with the microbubble gas.
 12. The method ofclaim 11, in which prior to converting into the powder, the film formingsurfactant is at least partially lamellarized.
 13. The method of claim12, in which prior to contacting withair or another gas the partiallylamellarized surfactant is admixed with the aqueous liquid carrier. 14.The method of claims 12 or 13, in which the liquid carrier furthercontains stabiliser compounds selected from hydrosoluble proteins,polypeptides, sugars, poly- and oligo-saccharides and hydrophilicpolymers.
 15. The method of claim 12, in which the conversion iseffected by coating the surfactant onto particles of soluble orinsoluble materials leaving the coated particles for a while under airor a gas, and admixing the coated particles with an aqueous liquidcarrier.
 16. The method of claim 12, in which the conversion is effectedby sonicating or homogenising under high pressure an aqueous solution offilm forming lipids, this operation leading, at least partly, to theformation of liposomes.
 17. The method of claim 16, in which prior tocontacting of at least partially lamellarized surfactant with air oranother gas the liposome containing solution is freeze-dried.
 18. Themethod of claims 16 and 17, in which the water solution of film forminglipids also contains viscosity enhancers or stabilisers selected fromhydrophilic polymers and carbohydrates in weight ratio relative to thelipids comprised between 10:1 and 1000:1.
 19. A method of preparation ofa suspension of air or gas filled microbubbles comprising a film formingsurfactant, a hydrophilic stabiliser and an aqueous liquid carrier,characterised by dissolving the film forming surfactant and thehydrophilic stabiliser in an organic solvent, freeze drying the solutionto form a dry powder, contacting the powder with air or another gas andadmixing said powder with the aqueous carrier.
 20. The method of claim19, in which the hydrophilic stabiliser is polyethylene glycol,polyvinyl pyrrolidone, polyvinyl alcohol, glycolic acid, malic acid ormaltol.
 21. The method of claim 19 or 20, in which the organic solventis tertiary butanol, 2-methyl-2-butanol or C₂Cl₄F₂.
 22. A method ofmaking an injectable suspension of gas-filled microbubbles according toclaim 1, which comprises suspending laminarized phospholipids, andoptionally other additives, in an aqueous carrier liquid, saidphospholipids having been in contact with said gas prior or after beingsuspended, under conditions such that a concentration of saidmicrobubbles sufficient to provide an echographic respose is formed inthe suspension, allowing a portion of said phospholipids to form astabilization layer around said bubbles and thereafter depleting thecarrier liquid of the excess of phospholipids not involved inmicrobubble stabilization.